Hall Regulators and Effectors of Small GTPases: Rho Family

1. Auflage 2006
ISBN: 978-0-08-047939-2
Verlag: Elsevier Science & Techn.
Format: PDF
Kopierschutz: 1 - PDF Watermark

E-Book, Englisch, Band Volume 406, 848 Seiten, Web PDF

Reihe: Methods in Enzymology

The Ras superfamily (>150 human members) encompasses Ras GTPases involved in cell proliferation, Rho GTPases involved in regulating the cytoskeleton, Rab GTPases involved in membrane targeting/fusion and a group of GTPases including Sar1, Arf, Arl and dynamin involved in vesicle budding/fission. These GTPases act as molecular switches and their activities are controlled by a large number of regulatory molecules that affect either GTP loading (guanine nucleotide exchange factors or GEFs) or GTP hydrolysis (GTPase activating proteins or GAPs). In their active state, they interact with a continually increasing, functionally complex array of downstream effectors. Since the last Methods in Enzymology volume on this topic in 2000, Rho GTPases have continued to receive a huge amount of attention. The human genome sequence has revealed the full extent of the Rho GEF and Rho GAP families (over 80 members for each) and the challenge of identifying the molecular interactions and cellular pathways influenced by each of these regulators is a daunting prospect. This new volume, Regulators and Effectors of Small GTPases: Rho Family, describes some of the methods currently being used to examine Rho family GTPase regulation at the biochemical and cellular level. - Describes the methods currently being used to examine Rho family GTPase regulation at the biochemical and cellular levels - Includes new imaging techniques that revolutionize the ability to visualize GTPase activities - Over 150 international contributors

Autoren/Hrsg.

Hall, Alan

Inhaltsverzeichnis

1;Cover;1
2;Copyright Page;5
3;Table of Contents;6
4;Contributors to Volume 406;12
5;Preface;20
6;Volumes in Series;22
7;Chapter 1: Purification and Biochemical Properties of Rac1, 2, 3 and the Splice Variant Rac1b;45
8;Chapter 2: Biochemical Analyses of the Wrch Atypical Rho Family GTPases;55
9;Chapter 3: Purification of P-Rex1 from Neutrophils and Nucleotide Exchange Assay;70
10;Chapter 4: In Vitro Guanine Nucleotide Exchange Activity of DHR-2/DOCKER/CZH2 Domains;85
11;Chapter 5: Biochemical Characterization of the Cool (Cloned-Out-of-Library)/Pix (Pak-Interactive Exchange Factor) Proteins ;102
12;Chapter 6: GEF and Glucosylation Assays on Liposome-Bound Rac;114
13;Chapter 7: Phosphorylation of RhoGDI by p21-Activated Kinase 1;124
14;Chapter 8: Purification of ARAP3 and Characterization of GAP Activities;135
15;Chapter 9: Regulation of RhoGAP Specificity by Phospholipids and Prenylation;148
16;Chapter 10: Purification and Activity of the Rho ADP-Ribosylating Binary C2/C3 Toxin;161
17;Chapter 11: Purification of TAT-C3 Exoenzyme;172
18;Chapter 12: Imaging and Photobleach Correction of Mero-CBD, Sensor of Endogenous Cdc42 Activation;184
19;Chapter 13: Cdc42 and PI(4,5)P2-Induced Actin Assembly in Xenopus Egg Extracts;200
20;Chapter 14: In Vitro Reconstitution of Cdc42-Mediated Actin Assembly Using Purified Components;218
21;Chapter 15: Biochemical Analysis of Mammalian Formin Effects on Actin Dynamics;234
22;Chapter 16: Formin Proteins: Purification and Measurement of Effects on Actin Assembly;259
23;Chapter 17: Purification and Kinase Assay of PKN;278
24;Chapter 18: Purification and Enzyme Activity of ACK1;294
25;Chapter 19: Direct Activation of Purified Phospholipase C Epsilon by RhoA Studied in Reconstituted Phospholipid Vesicles;304
26;Chapter 20: Regulation of PLCbeta Isoforms by Rac;316
27;Chapter 21: Biochemical Properties and Inhibitors of (N-)WASP;325
28;Chapter 22: The Use of GFP to Localize Rho GTPases in Living Cells;340
29;Chapter 23: Analysis of the Spatiotemporal Activation of Rho GTPases Using Raichu Probes;359
30;Chapter 24: Measurement of Activity of Rho GTPases During Mitosis;376
31;Chapter 25: Inhibition of Rho GTPases by RNA Interference;389
32;Chapter 26: RNA Interference Techniques to Study Epithelial.Cell Adhesion and Polarity;406
33;Chapter 27: Nucleofection of Primary Neurons;418
34;Chapter 28: Dock180-ELMO Cooperation in Rac Activation;432
35;Chapter 29: Rho GTPase Activation by Cell-Cell Adhesion;446
36;Chapter 30: Activation of Rap1, Cdc42, and Rac by Nectin Adhesion System;459
37;Chapter 31: Analysis of Activated GAPs and GEFs in Cell Lysates;469
38;Chapter 32: Degradation of RhoA by Smurf1 Ubiquitin Ligase;481
39;Chapter 33: Ubiquitin-Mediated Proteasomal Degradation of Rho Proteins by the CNF1 Toxin;491
40;Chapter 34: Regulation of Superoxide-Producing NADPH Oxidases in Nonphagocytic Cells;500
41;Chapter 35: Activation of MEKK1 by Rho GTPases;512
42;Chapter 36: Activation of the Apoptotic JNK Pathway Through the Rac1-Binding Scaffold Protein POSH;523
43;Chapter 37: Quantification of Isozyme-Specific Activation of Phospholipase C-beta2 by Rac GTPases and Phospholipase C-epsiv by Rho GTPases in an Intact Cell Assay System;533
44;Chapter 38: Activation of Rho and Rac by Wnt/Frizzled Signaling;544
45;Chapter 39: Fluorescent Assay of Cell-Permeable C3 Transferase Activity;556
46;Chapter 40: Use of TIRF Microscopy to Visualize Actin and Microtubules in Migrating Cells;564
47;Chapter 41: Inhibition of ROCK by RhoE;577
48;Chapter 42: Conditional Regulation of a ROCK-Estrogen Receptor Fusion Protein;585
49;Chapter 43: Rational Design and Applications of a Rac GTPase-Specific Small Molecule Inhibitor;598
50;Chapter 44: In Vitro Assay of Primary Astrocyte Migration as a Tool to Study Rho GTPase Function in Cell Polarization;609
51;Chapter 45: Real-Time Centrosome Reorientation During Fibroblast Migration;623
52;Chapter 46: Lentiviral Delivery of RNAi in Hippocampal Neurons;637
53;Chapter 47: Methods for Studying Neutrophil Chemotaxis;649
54;Chapter 48: Measurement of Epidermal Growth Factor Receptor Turnover and Effects of Cdc42;658
55;Chapter 49: Tumor Cell Migration in Three Dimensions;669
56;Chapter 50: An In Vitro Model to Study the Role of Endothelial Rho GTPases During Leukocyte Transendothelial Migration;687
57;Chapter 51: Analysis of a Mitotic Role of Cdc42;700
58;Chapter 52: Plexin-Induced Collapse Assay in COS Cells;709
59;Chapter 53: Morphological and Biochemical Analysis of Rac1 in Three-Dimensional Epithelial Cell Cultures;720
60;Chapter 54: Using Three-Dimensional Acinar Structures for Molecular and Cell Biological Assays;736
61;Chapter 55: TC10 and Insulin-Stimulated Glucose Transport;745
62;Chapter 56: GTPases and the Control of Neuronal Polarity;759
63;Chapter 57: In Vitro Assembly of Filopodia-Like Bundles;771
64;Author Index;785
65;Subject Index;821